A multitude of laboratory immunoassay tests for analytes of interest are performed on biological samples for diagnosis, screening, disease staging, forensic analysis, pregnancy testing, drug testing, and other reasons. While a few qualitative tests, such as pregnancy tests, have been reduced to simple kits for the patient's home use, the majority of quantitative tests still require the expertise of trained technicians in a laboratory setting using sophisticated instruments. Laboratory testing increases the cost of analysis and delays the results. In many circumstances, delay can be detrimental to a patient's condition or prognosis, such as for example the analysis of markers indicating myocardial infarction and heart failure. In these and similar critical situations, it is advantageous to perform such analyses at the point-of-care, accurately, inexpensively, and with a minimum of delay.
Two-site immunoassays, also called sandwich-type immunoassays, are often employed for determining analyte concentration in biological test samples, and are used, for example, in the point-of-care analyte detection system developed by Abbott Point-of-care Inc. as the i-Stat® system. In a typical two-site enzyme-linked immunosorbent assay (ELISA), one antibody is bound to a solid support to form an “immobilized antibody” and a second antibody is conjugated or bound to a signal-generating reagent such as an enzyme to form a “signal antibody.” Upon reaction with a sample containing the analyte to be measured, the analyte becomes “sandwiched” between the immobilized antibody and the signal antibody. After washing away the sample and any non-specifically bound reagents, the amount of signal antibody remaining on the solid support is measured and should be proportional to the amount of analyte in the sample.
Many types of immunoassay devices and processes have been described. One disposable sensing device for successfully measuring analytes in a sample of blood is disclosed by Lauks in U.S. Pat. No. 5,096,669. Other devices are disclosed by Davis et al. in U.S. Pat. Nos. 5,628,961 and 5,447,440 for a clotting time. These devices employ a reading apparatus and a cartridge that fits into the reading apparatus for the purpose of measuring analyte concentrations and viscosity changes in a sample of blood as a function of time. U.S. Pat. Nos. 5,096,669; 5,628,961 and 5,447,440 are hereby incorporated herein by reference in their entireties.
Electrochemical detection, in which binding of an analyte directly or indirectly causes a change in the activity of an electroactive species adjacent to an electrode, has also been applied to immunoassay. For a review of electrochemical immunoassay, see Laurell et al., Methods in Enzymology, vol. 73, “Electroimmunoassay”, Academic Press, New York, 339, 340, 346-348 (1981).
Microfabrication techniques (e.g. photolithography and plasma deposition) are attractive for construction of multilayered sensor structures in confined spaces. Methods for microfabrication of electrochemical immunosensors, for example on silicon substrates, are disclosed in U.S. Pat. No. 5,200,051 to Cozzette et al., which is hereby incorporated by reference in its entirety. These include dispensing methods, methods for attaching biological reagent, e.g. antibodies, to surfaces including photoformed layers and microparticle latexes, and methods for performing electrochemical assays.
In an electrochemical immunosensor, the binding of an analyte to its cognate antibody produces a change in the activity of an electroactive species at an electrode that is poised at a suitable electrochemical potential to cause oxidation or reduction of the electroactive species. There are many arrangements for meeting these conditions. For example, electroactive species may be attached directly to an analyte, or the antibody may be covalently attached to an enzyme that either produces an electroactive species from an electroinactive substrate, or destroys an electroactive substrate. See, M. J. Green (1987) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 316:135-142, for a review of electrochemical immunosensors.
The concept of differential amperometric measurement is well known in the electrochemical art, see for example jointly owned Cozzette, U.S. Pat. No. 5,112,455. In addition, a version of a differential amperometric sensor combination is disclosed in jointly owned Cozzette, U.S. Pat. No. 5,063,081. This patent also discloses the use of permselective layers for electrochemical sensors and the use of film-forming latexes for immobilization of bioactive molecules, incorporated here by reference. The use of poly(vinyl alcohol) (PVA) in sensor manufacture is described in U.S. Pat. No. 6,030,827 incorporated here by reference. Vikholm (U.S. 2003/0059954A1) teaches antibodies directly attached to a surface with a biomolecule repellant coating, e.g. PVA, the surface in the gaps between antibodies, and Johansson (U.S. Pat. No. 5,656,504) teaches a solid phase, e.g. PVA, with antibodies immobilized thereon. U.S. Pat. Nos. 6,030,827 and 6,379,883 teach methods for patterning poly(vinylalcohol) layers and are incorporated by reference in their entirety.
US 20060160164 describes an immunoassay device with an immuno-reference electrode, US 20050054078 describes an immunoassay device with improved sample closure, US 20040018577 describes a multiple hybrid immunoassay, and US 20030170881 (issued as U.S. Pat. No. 7,419,821) describes an apparatus and methods for analyte measurement and immunoassay, all of which are jointly owned and are incorporated here by reference.
With regard to amperometric measurements, there are several means known in the art for reducing the importance of the non-Faradaic component of the signal, thus increasing sensitivity. These include newer electrochemical methods, e.g. using square wave voltammetry in place of chronoamperometry, and chemical means, e.g. an alkyl thiol reagent to passivate an electrode surface.
One limitation of conventional assay configurations, however, is the susceptibility to interference caused by heterophile antibodies that may be present in the test sample. See, e.g., L. Kricka, “Human Anti-Animal Antibody Interferences in Immunological Assays,” Clinical Chemistry 45:7 at 942-956 (1999). Antibodies employed in commercial immunoassays are in many cases prepared or “raised” in animals or media of animal origin. Furthermore, many individuals harbor naturally occurring, non-specific antibodies to animal proteins, “endogenous antibodies,” that may bind to the animal antibody reagents employed in the immunoassay, leading to erroneous results. For example, endogenous antibodies capable of binding to one or more of the assay reagents pose the potential to generate erroneous test results by cross-linking the reagents, leading to false-positive results, or sequestering the reagents, leading to false-negative results.
It has been found, for example, that cancer therapy with radiolabelled murine monoclonal antibodies can lead to the production of human anti-mouse antibodies (HAMA) in the patient. It was subsequently shown that the presence of HAMA in serum samples taken from those patients, can cause cross-linking of the reagent murine monoclonal antibodies used in sandwich-type enzyme immunoassays for cancer markers (Boscato et al., Heterophile antibodies: A problem for all immunoassays, Clin Chem 34, 27-33, 1988). In addition, Nicholson et al., (Immunoglobulin inhibiting reagent (IIR): Evaluation of a new method for eliminating spurious elevation in CA125 caused by HAMA, Intl J Biol Markers 11, 46-49, 1996) demonstrated beneficial results using IIR (Bioreclamation Inc, NY) to eliminate HAMA interference in a CA125 assay. The IIR material is reported to comprise a partially purified preparation of immunoglobulins (IgG, IgM) from several species, principally murine IgG (subtypes IgG2a, IgG2b and IgG3) from Balb/c mice.
U.S. Pat. No. 6,106,779 teaches that nonspecific binding of certain assay reagents to each other and to device components is often a problem in diagnostic assays. This is particularly a problem when an antibody recognizes a region of a molecule that is not its antigen. This can then lead to high background reactions and false positive (or negative) assay results. Non-specific binding inhibitors that may be used for this problem include bovine IgG.
US 20080261242 discusses endogenous human heterophile antibodies and human anti-animal antibodies, which have the ability to bind to immunoglobulins of other species, and are present in the serum or plasma of more than 10% of patients. These circulating heterophile antibodies may interfere with immunoassay measurements. In sandwich immunoassays, these heterophile antibodies can either bridge the capture and detection (diagnostic) antibodies, thereby producing a false-positive signal, or they may block the binding of the diagnostic antibodies, thereby producing a false-negative signal. Additionally, in competitive immunoassays, the heterophile antibodies may bind to the analytic antibody and inhibit its binding to the analyte. They also may either block or augment the separation of the antibody-analyte complex from free analyte, especially when anti-species antibodies are used in separation systems. As a result, the impact of these heterophile antibody interferences are often difficult to predict.
Several additional methods for removing heterophile antibodies from samples are also known and include: (i) heating the specimen in a sodium acetate buffer, pH 5.0, for 15 minutes at 90 degrees C. followed by centrifuging at 1200 g for 10 minutes, (ii) precipitation using polyethylene glycol (PEG), and (iii) immunoextraction with protein A or protein G. Clinical guidelines for dealing with the heterophile antibody issue are also provided by the Clinical and Laboratory Standards Institute (CLSI) Immunoassay Interference by Endogenous Antibodies; Proposed Guideline. CLSI document I/LA30-P (ISBN 1-56238-633-6).
Generally, immunoassay manufacturers strive to reduce heterophile interference by (a) removal or inactivation of the interfering immunoglobulins from samples, (b) modification of assay antibodies to make them less prone to react with heterophile antibodies, and (c) use of blocking agents (mostly IgGs) that reduce interference.
However, the need remains for improved processes for ameliorating heterophile antibodies in at least the following areas: (i) immunosensor interference, most notably in the context of point-of-care testing, (ii) electrochemical immunoassays, (iii) the use of an immunosensor in conjunction with an immuno-reference sensor, (iv) whole blood immunoassays, (v) single-use cartridge based immunoassays, (vi) non-sequential immunoassays with only a single wash step, and (vii) dry reagent coatings.